Additives to tampons

ABSTRACT

Absorbent products, especially catamenial tampons, for absorbing body fluids, such as menstrual fluid, blood and wound exudates, comprise an amount of a compound effective to inhibit the production of toxic shock syndrome toxin-1 by Staphylococcus aureus bacteria when the products are brought into contact with the bacteria. The compound is selected from the group consisting of monoesters of a polyhydric aliphatic alcohol and a C 8  -C 18  fatty acid; diesters of a polyhydric aliphatic alcohol and a C 8  -C 18  fatty acid; and mixtures thereof. The monoesters and diesters have at least one hydroxyl group associated with their aliphatic alcohol residue.

This is a continuation of application Ser. No. 08/183,446, filed Jan.14, 1994, now abandoned, which is a Continuation of Ser. No.08/041,134,filed Mar. 29, 1993, now abandoned, which is a Continuationof Ser. No. 07/864,704, filed Apr. 7, 1992, now abandoned, which is aContinuation of Ser. No. 07/695,358, filed May 3, 1991, now abandoned,which is a Continuation of Ser. No. 07/508,521, filed Apr. 17, 1990, nowabandoned, which is a Continuation of Ser. No. 07/343,965, filed Apr.27, 1989, now abandoned.

FIELD OF THE INVENTION

The present invention relates to absorbent products and especially toabsorbent products such as tampons, sanitary napkins, wound dressingsand the like which are adapted to absorb body fluids like menstrualfluid, blood and wound exudates. More particularly, the inventionrelates to catamenial tampons which, owing to the presence therein orthereon of certain inhibitory agents, reduce the amount of toxinsproduced by bacteria coming into contact therewith.

BACKGROUND OF THE INVENTION

Menstrually occurring toxic shock syndrome (TSS), a severe and sometimesfatal multi-system disease associated with infection or colonization byStaphylococcus aureus (S. aureus) bacteria, has been linked to the useof tampons during menstruation. The disease is believed to be caused bytoxic shock syndrome toxin-1 (TSST-1), the toxin produced by themajority of Staphlococcal strains isolated from menstrual TSS patients.

Subsequent to the publication of reports associating toxic shocksyndrome with the use of tampons, a number of investigators undertookstudies designed to evaluate the effect of tampons on growth of S.aureus bacteria as well as the effect of tampons on the production ofTSST-1 by that bacteria. Early efforts to elucidate the role of tamponsin TSS yielded conflicting data. Schlievert et al. (Obstet. Gynecol.,Vol. 64, pp. 666-670, November 1984) studied the effect of tampons on S.aureus to evaluate whether or not tampon components increase growth ofS. aureus and production of toxic shock syndrome toxin-1. It wasconcluded that, under the test conditions of their study, tamponcomponents provide neither nutrients for growth of toxic shock syndromeS. aureus nor factors that induce production of toxic shock syndrometoxin-1 above control levels. After six hour incubation, somecommercially available tampons which were tested were inhibitory tobacterial growth and suppressed toxin production. Others suppressedtoxin production but did not inhibit cell growth. One tampon inhibitedcell growth but increased the amount of toxin produced. On the otherhand, Tierno and Hanna (Contraception, Vol. 31, pp 185-194, 1985)reported that in their experiments tampons did stimulate S. aureus toproduce TSST-1.

Reiser et al. (J. Clin. Microbiol., Vol. 25, No. 8, pp 1450-1452, August1987) thereafter reported the results of tests they conducted todetermine the effect of four brands of tampons on production of toxicshock syndrome toxin-1. The amount of air available to the tampons whichwere tested was limited to that contained in sacs (made from cellulosesausage casing with a molecular weight cut-off of less than 10,000) inwhich the tampons were enclosed during testing. This method was deemedadvantageous in that the limited amount of available air was thought tomimic more closely than previously used methods the in vivo condition inthe vagina during menstruation with a tampon in place and in that thetampons which were tested were not altered prior to testing. The resultsof the tests conducted by Reiser et al. indicated that tampons provideincreased surface area for the S. aureus bacteria to grow and adequateoxygen for toxin production. No significant inhibition of growth of thestaphylococci bacteria or TSST-1 production by any of the tampons testedwas noted.

Robbins et al., publishing in J. Clinical Microbiol., Vol. 25, No. 8,pp. 1446-1449, August 1987 at the same time as Reiser et al., reportedthe effect of 17 commercially available tampons on TSST-1 toxinproduction using a disk-membrane-agar (DMA) method, with incubation at37° C. for 19 hours under 5% CO₂ in air. Filter membranes overlayingagar medium (with or without blood) in small petri dishes were spreadinoculated with a TSST-1 producing strain of S. aureus. Robbins et al.concluded that the main role of tampons in TSS may be that of providinga fibrous surface for heavy colonization and sufficient air for TSST-1production. In addition, they found evidence of inhibition of TSST-1production by additives such as the deodorant/surfactant used in acommercially available deodorant tampon and a decrease in TSST-1production by inhibiting growth of S. aureus as was observed in the caseof a different commercially available tampon. It was thought that bothinhibition of TSST-1 production and inhibition of S. aureus growth mightprove to be important in reducing the risk of TSS.

U.S. Pat. No. 4,405,323 to Auerbach discloses a tampon designed toeliminate the hazards of toxic shock syndrome and dysmenorrhea. Thetampon has incorporated therein an antibacterial agent which is said todisperse on contact with body fluids and prevent development of theorganisms which produce the toxins which cause toxic shock syndrome.Among the antibacterial materials disclosed for use are povidone-iodinecompound, mercury, zinc, penicillin, erythromycin and nitrofurazone.

Patent Cooperation Treaty Publication No. WO 86/05388 (published Sep.25, 1986) to Kass teaches that the inclusion of a salt of a nontoxicdivalent cation in absorptive pads, e.g. catamenial tampons, inhibitsproduction of toxic shock syndrome toxin-1 and other staphylococcalproducts during use of said absorptive pad. Suitable salts include thoseof magnesium, barium, calcium or strontium (preferred) or of otherdivalent cations such as zinc, manganese, copper, iron, nickel and thelike. The anionic portion of the salt is not critical. Magnesiumstearate and magnesium acetate are particularly preferred salts for usein the invention.

In U.S. Pat. No. 4,374,522 to Olevsky it is stated that patterns of useof catamenial tampon seem to indicate that high absorptive capacity withthe concomitant extended period of use of certain tampons are factorswhich contribute to the formation of toxic shock syndrome. The inventiontheorizes that tampons having limited absorptive capacity and requiringrelatively more frequent changes may be desirable. The Olevsky patentprovides a tampon made of conventional cellulosic materials, such asrayon fibers, which have been compressed into a bullet-shape with anopen bottom surface sealed by a fluid impermeable sheet. The fluidimpermeable bottom and the traditional bullet shaped pledger define ahollow core central reservoir area which is said to serve as a reservoirfor excess menstrual fluid.

U.S. Pat. No. 4,431,427 to Lefren et al. discloses menstrual tamponscomprising physiologically safe, water-soluble acids in their monomeric,oligomeric or polymeric forms. Citric, glycolic, malic, tartaric andlactic acids are disclosed as being useful in the practice of theinvention. The presence of one or more of the above-noted acids in atampon is said to inhibit the growth of bacteria responsible for toxicshock. Where an acid is used in its polymeric form, the tampon mayadditionally include an enzyme to hydrolyze the polymeric acid to itsmonomeric form.

Canadian Patent No. 1,123,155 to Sipos discloses a catamenial tampon forpreventing toxic shock syndrome during menstrual flow. The body of thetampon, which is open at the insertion end and is closed at thewithdrawal end, is snugly surrounded in its expanded condition by afluid proof, thin and flexible membrane. This membrane, which can bemade of polyethylene sheet, is biased against the vaginal wall duringuse of the tampon, is neutral to the vaginal mucosa and is completelyimpermeable to bacteria, viruses and toxic decomposition products of themenstrual flow.

Canadian Patent No. 1,192,701 to Bardhan discloses a tampon for theabsorption of menstrual flow and comprising an inner layer ofliquid-absorbent material and an outer layer which surrounds andencloses the inner layer. Menstrual discharge may flow inwardly to theinner layer but the outer layer is impervious to the passage ofmenstrual fluid outwardly from the inner layer. A plurality of liquidabsorbent wicks extending from the inner layer through apertures formedin the outer layer serve as conduits for the flow of menstrual dischargefrom outside the tampon to the inner layer thereof. The disclosedstructure is said to minimize the availability of discharge outside thetampon with a resulting reduction in the likelihood of growth of S.aureus and consequently its production of toxin. This patent alsodiscloses that an antimicrobial compound which is bactericidal orbacteriostatic to S. aureus may be included in the inner layer. Theantimicrobial agent may take the form of an antibiotic (such aspenicillin, erythromycin, tetracycline or neomycin), a chemotherapeuticagent (such as a sulfonamide) or a disinfectant (such as phenol). Thepatent states that since the tampon is protected by its outer layer fromcontact with the vaginal wall, the risk of an allergic or other adversereaction to the anti-microbial agent is minimized, and since theantimicrobial agent is also protected by the outer layer from contactwith menstrual discharge, there is little risk of the destruction ofcommensal organisms in the vagina or development of resistance to theantimicrobial agent by S. aureus in any menstrual discharge outside thevagina.

S. Notermans et al. (Journal of Food Safety, Vol. 3 (1981), pages 83-88)reported that glyceryl monolaurate, when used in the proportion of 5 gper kg. of meat slurry (pH 6.0-6.2) inhibited toxin. productions byClostridium botulinum type A, type B and type E. This article does notmention Staphylococcus aureus nor any toxin(s) produced therefrom nordoes it mention absorbent products or toxic shock syndrome.

U.S. Pat. No. 4,585,792 to Jacob et al. discloses that L-ascorbic acidwhen topically applied to the vaginal area of a human female duringmanses will inactivate toxins known to contribute to Toxic ShockSyndrome. The ascorbic acid compound may be carried by a vaginal tampon.The disclosure of U.S. Pat. No. 4,722,937, is to the same effect.

U.S. Pat. No. 4,413,986 to Jacobs discloses a sterilely-packaged tamponassembly for sterile insertion of a tampon into the vagina and having aguide tube telescoped around an insertion tube and a flexible sheathattached to the inner end of the guide tube and tucked into the innerend of the insertion tube. In use, as the insertion tube is pushedthrough the guide tube and into the vagina, the flexible sheath ispulled over the inner end of the insertion tube and extends along theexterior thereof. The portion of the insertion tube which is insertedinto the vagina is at all times fully sheathed by the flexible sheath.

SUMMARY OF THE INVENTION

In accordance with the present invention, it has now been discoveredthat an absorbent product comprising a compound selected from the groupconsisting of:

a) a monoester of a polyhydric aliphatic alcohol and a fatty acidcontaining from eight to eighteen carbon atoms and wherein saidmonoester has at least one hydroxyl group associated with its aliphaticalcohol residue;

b) diesters of a polyhydric aliphatic alcohol and a fatty acidcontaining from eight to eighteen carbon atoms and wherein said diesterhas at least one hydroxyl group associated with its aliphatic alcoholresidue; and

c) mixtures of the aforesaid monoesters and diesters unexpectedlyreduces the amount of toxic shock syndrome toxin-1 produced in vitrowhen said absorbent product is exposed to Staphylococcus aureusbacteria.

The fatty acid portion of the aforementioned monoesters and diesters maybe derived from caprylic, captic, lauric, myristic, palmitic and stearicacids, which are saturated fatty acids whose chain lengths,respectively, are C₈, C₁₀, C₁₂, C₁₄, C₁₆ and C₁₈. The fatty acid portionof the aforementioned monoesters and diesters may be derived as wellfrom unsaturated fatty acids having carbon chain lengths also rangingfrom C₈ to C₁₈, one example of such unsaturated fatty acids being oleicacid. The preferred fatty acid for use in the practice of the presentinvention is lauric acid, a saturated fatty acid whose chemical formulais C₁₁ H₂₃ COOH.

As used in this specification and the appended claims, the term"aliphatic" has the meaning usually accorded it in organic chemistry,i.e. "aliphatic" refers to organic compounds characterized bystraight--or branched--chain arrangement of the constituent carbonatoms.

As used in this specification and the appended claims, the term"polyhydric" refers to the presence in a chemical compound of at leasttwo hydroxyl (OH) groups. Thus, a polyhydric aliphatic alcohol is onewhich has at least two hydroxyl groups and in which the carbon backboneis either straight or branched.

Polyhydric alcohols suitable for forming monoesters and/or diesters foruse in the practice of the present invention are 1,2-ethanediol;1,2,3-propanetriol (glycerol); 1,3-propanediol; 1,4-butanediol;1,2,4-butanetriol and the like. The preferred polyhydric aliphaticalcohol for forming monoesters and diesters for use in the practice ofthe present invention is 1,2,3-propanetriol (commonly called glycerol)whose formula is HOCH₂ CH(OH)CH₂ OH.

It will be observed that the esters which are useful in the practice ofthe present invention have at least one hydroxyl group associated withtheir aliphatic alcohol residue. Thus, it will be understood that themonoester of 1,2-ethanediol and one of the aforementioned fatty acidsmay be used in the practice of the present invention because said ester,whose general formula is ##STR1## has at least one hydroxyl group (i.e.the hydroxyl group at the far right-hand side of the structural formulashown above) in that portion of the ester derived from the aliphaticalcohol 1,2-ethanediol. On the other hand, it will be understood thatthe diester of 1,2-ethanediol and one of the aforementioned fatty acidscannot be used in the practice of the present invention because saidester, whose general formula is ##STR2## does not have at least onehydroxyl group in that portion of the ester derived from the1,2-ethanediol.

The monoester of glycerol and one of the designated fatty acids may beused in the practice of the present invention because that ester willhave two hydroxyl groups associated therewith which are derived from theglycerol. The diester of glycerol and one of the designated fatty acidsmay also be used because that ester will have one hydroxyl groupassociated therewith which is derived from the aliphatic alcoholglycerol. Indeed, as will be seen hereinafter, blends of glycerolmonolaurate and glycerol dilaurate have been found to be useful in thepractice of the present invention. Finally, it will be understood thatthe triester of glycerol and one of the designated fatty acids cannot beused in the practice of the present invention because that ester doesnot have at least one hydroxyl group in that portion thereof which isderived from the aliphatic alcohol, i.e. glycerol.

Preferred esters for use in the practice of the present invention areglyceryl monolaurate, glyceryl dilaurate and mixtures thereof.

In accordance with the invention, the absorbent product contains anamount of the above-described ester which is effective to inhibit theformation of TSS toxin-1 when said product is exposed to S. aureus. Forexample, effective amounts have been found to be from about 0.1% andhigher and, preferably, at least about 0.5% of the specified mono- ordiester compound (or mixtures thereof), based on the weight of theabsorbent material comprising the absorbent product. As used herein, theterm "absorbent material" includes natural or synthetic fibers, films,foams, wood pulp, peat moss, superabsorbent polymers and the like whichare capable, either inherently or by virtue of the manner in which theyhave been assembled, of absorbing liquids such as water, urine,menstrual fluids, blood, wound exudates and the like.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates the growth curves of cultures with and without one ofthe toxin-inhibiting compounds of the present invention.

FIGS. 2A-C illustrate the results of Northern blot analysis of cellcultures.

FIGS. 3A and 3B illustrate graphs of transcriptional fusion of TSST-1promoter sequence to the structural gene encoding beta-lactamaseactivity.

FIGS. 4A and 4B illustrate graphs showing inhibition of alpha hemolysinactivity by one of the compounds of the present invention.

FIGS. 5A and 5B illustrate graphs confirming inhibition of alphahemolysin activity by one of the compounds of the present inventionusing transcription analysis.

GENERAL PROCEDURE FOR PREPARING TAMPONS OF THE INVENTION

During the course of conducting the investigations forming the basis forthe present patent application, varying amounts of the aforementionedester compounds (or mixtures thereof) were added to several differentkinds of tampons. These tampons included ones prepared in thelaboratories of the assignee of the present patent application as wellas commercially available tampons made by several differentmanufacturers. Tampons of one manufacturer had different weights fromthose of another manufacturer and, indeed, no two tampons from a givenmanufacturer had identical weights. The ester compounds to beinvestigated were dissolved in isopropyl alcohol to form solutions whichwere then uniformly applied, by pipetting, to the outer surfaces of thevarious tampons, after which the isopropyl alcohol was evaporated toprovide a tampon comprising the ester compound.

In order to ensure that the absorptive capacity of the tampons was notexceeded, it was decided to fix the amount of isopropyl alcohol solutionapplied to each tampon at four (4) grams in all cases. In view of thisdesire to hold constant the weight of isopropyl alcohol solution appliedto each tampon, it was necessary to vary the concentration of estercompound in the isopropyl alcohol solution in order to vary the level ofthe selected ester compounds in the tampon. Accordingly, the followinggeneral procedure was used to apply a given ester material to a tampon.

Tampons were labeled for identification and weighed to the nearestone-tenth of a gram. The amount of ester required to give the desiredconcentration in the treated tampon was then calculated. Solutions ofthe ester in reagent grade isopropyl alcohol were prepared atconcentrations such that four (4) grams of the solution contained theamount of ester to be included in the tampon to be tested. In thismanner, the amount of ester in a given tampon could be varied while thetotal weight of the ester/isopropyl alcohol solution used to prepareeach individual tampon was held constant at four (4) grams. For example,if the untreated tampon weighed 2.6 grams, then 0.26 gram of ester wasrequired to provide a tampon comprising 10% of the ester based on theweight of the untreated tampon (i.e. 2.6 gram tampon weight×0.10=0.26gram ester). In this instance, six and one-half (6.5) grams of esterwere dissolved in ninety-three and one-half (93.5) grams of reagentgrade isopropyl alcohol to give a solution containing six and one-half(6.5%) by weight of ester. Four (4) grams of this solution contained therequired 0.26 gram of ester. As another example, if the untreated tamponweighed 2.8 grams, and it was desired that the concentration of ester beone percent (1%) based on the weight of the untreated tampon, a solutioncontaining 0.70 grams of ester and 99.3 grams of isopropyl alcohol wasprepared. Four grams of this solution then contained the required 0.028grams of ester.

As a third example, if the untreated tampon weighed 2.5 grams, and itwas desired that the concentration of ester be 0.1% based on the weightof the untreated tampon, a solution containing 0.0625% by weight ofester in isopropyl alcohol was prepared. Four (4) grams of this solutioncontained the required 0.0025 gram of ester.

All solutions of ester compound in isopropyl alcohol were thoroughlystirred to ensure uniformity. In addition, especially at higherconcentrations, the rate of dissolution of the ester could be increasedby warming the ingredients to about 60° C., e.g. in a heated water bath.

Once the tampon had been weighed and the appropriate solution of esterin reagent grade isopropyl alcohol had been prepared in the mannerexplained above, four (4) grams of the ester/isopropyl alcohol solution(at room temperature) were uniformly applied, by pipetting, to the outersurfaces of the tampon. Tampons were rotated during the application ofthe ester solution to ensure as uniform an application as possible. Theisopropyl alcohol was then evaporated at 70° C. in a hooded drying ovento yield a tampon comprising the desired level of ester compound.

The foregoing procedure was used to prepare all the ester-containingtampons mentioned in the Examples of the present patent application.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In Example 1 which follows herein, the invention will be described indetail in connection with a catamenial tampon comprising an absorbentmaterial, a liquid-pervious covering fabric, and an amount of a mixtureof glycerol monolaurate and glycerol dilaurate which is effective toinhibit the production of toxic shock syndrome toxin-1 by S. aureusbacteria when said bacteria are brought into contact with the tampon. Itwill be understood that the principles of the invention apply as well toother absorbent products such as wound dressings, disposable diapers,sanitary napkins and other kinds of tampons, such as those intended formedical, surgical, dental and/or nasal use.

Catamenial tampons comprising rayon fibers as their absorbent materialwere prepared as follows. The rayon fibers employed were 3-denier,vicose rayon staple fibers having a length of 11/8 inches (2.86 cm) and11-25 crimps per inch (about 4.3-9.8 crimps per centimeter). The fiberswere 100% vicose rayon, i.e. they were substantially free of allfinishes and additives, such as surfactants and the like, commonly usedin commercial production.

Using commercially available carding equipment, the above-describedrayon fibers were carded into a fibrous web weighing about 520grains/yd² (33.6 grams/meter²). The carded web of rayon fibers wasgathered into a tubular ribbon having a diameter of about one-inch (2.54cm). This tubular ribbon was thereafter covered in a nonwoven fabricmade from heat-fusible fibers and weighing about 0.25 oz/yd² (7.08gm/m²). The edges of the heat-fusible nonwoven fabric were overlappedslightly and subsequently heat treated to form a seal. The coveredribbon of rayon fibers was cut into blanks. A white rayon string waspierced and looped through each blank. The blank was then compressed inknown fashion to provide a test tampon having a diameter of 0.47 inch(1.2 cm), a length of 1.75 inches (4.44 cm), and a weight of about 2.6grams. The dangling portion of the withdrawal string was cut from thetampon prior to testing.

A mixture of glycerol monolaurate and glycerol dilaurate, commerciallyavailable under the tradename "Lauricidin", was obtained fromLauricidin, Inc. located in East Lansing, Mich., U.S.A. This mixture,which is hereinafter sometimes referred to as "Lauricidin", was analyzedand found to contain 93 percent by weight of glycerol monolaurate and3.5 percent by weight of glycerol dilaurate. It is known that Lauricidinhas antimicrobial properties and is non-toxic to humans. It has beensuggested for use in anti-caries products, insecticides, cosmeticpreparations and food compositions.

EXAMPLE 1

Tampons comprising, respectively, 0.1%, 1.0% and 10% by weight of theaforementioned Lauricidin mixture based on the weight of the untreatedtampon were prepared using the above-mentioned test tampons. TheLauricidin mixture was applied to the test tampons according to theGeneral Procedure for Preparing Tampons of the Invention describedearlier in this application. The Lauricidin containing tampons wereprepared in duplicate. The Lauricidin treated tampons were then testedaccording to the Tampon Sac Method reported by Reiser et al. in theJournal of Clinical Microbiology, Vol. 25, August 1987, pp. 1450-1452,the disclosure of which is hereby incorporated by reference.Staphylococcus aureus strain FRI-1169, obtained in lyophilized form fromDr. Merlin Bergdoll, Food Research Institute, University of Wisconsin,in Madison, Wisc., U.S.A., was employed in the tests. A S. aureussuspension was prepared by thoroughly mixing one (1) milligram of thelyophilized S. aureus strain to one (1) milliliter of Brain HeartInfusion (BHI) Broth (obtained from Difco Laboratories, Detroit, Mich.,U.S.A.), transferring said mixture into a test tube containing five (5)milliliters of BHI Broth, thoroughly mixing again, and incubating fortwenty-four (24) hours at 37° C. prior to use.

100 milliliters of brain heart infusion (BHI) agar (also obtained fromDifco Laboratories in Detroit, Mich., U.S.A.) were put into each of ten3.8 cm×20 cm culture tubes. Cellulose sacs were made and sterilized inthe manner reported by Reiser et al. The sterile cellulose sacs wereinoculated with the aforementioned S. aureus suspension in an amountsufficient to provide at the beginning of the test a concentrationtherein of 1×10⁸ CFU/ml Staphylococcus aureus bacteria.

Each Lauricidin treated tampon to be tested was inserted into a sterilecellulose sac containing the S. aureus bacteria and each sac was theninserted into a culture tube containing the BHI agar. Two controls, eachin duplicate, were used. In one control (called the "inoculum control"),an inoculated sac (with no tampon therein) was placed in each of twoculture tubes containing BHI agar. In the second control, two untreatedtampons (i.e. tampons made in the described manner but with noLauricidin in the isopropyl alcohol) were placed in cellulose sacs whichin turn were placed in culture tubes containing BHI agar. Thus, tenculture tubes were used in this test, four containing the aforementionedcontrols (two with tampons; two without tampons) and the otherscontaining the aforementioned Lauricidin-treated test tampons induplicate.

The concentrations of S. aureus strain FRI-1169 and toxic shock syndrometoxin-1 at the outset of the test (0 hours) and after incubation for 24hours at 37° C. are shown in Table 1.

                                      TABLE 1                                     __________________________________________________________________________    THE EFFECT OF LAURICIDIN TREATED TAMPONS ON TSST-1 FORMATION                  BY AND GROWTH OF STAPHYLOCOCCUS AUREUS                                               INITIAL               FINAL                                                   CONCENTRATION                                                                            INITIAL    CONCENTRATION                                                                             FINAL      FINAL                            OF S. AUREUS                                                                             CONCENTRATION                                                                            OF S. AUREUS                                                                              CONCENTRATION                                                                            CONCENTRATION                    CELLS      OF         CELLS       OF S. AUREUS                                                                             OF                               (CFU/ml)   TSST-1.sup.a)                                                                            (CFU/ml)    CELLS.sup.b)                                                                             TSST-1.sup.a              SAMPLE (10.sup.8) (ng/ml)    (10.sup.8)  (/ml)      ug/ml                     __________________________________________________________________________    No tampon                                                                            1.0        ND         100         10.0       50                        (control)                                                                     Untreated                                                                            1.0        ND         100         10.0       31                        Tampon                                                                        (control)                                                                     Tampon 1.0        ND         794         10.9       15                        w/0.1%                                                                        Lauricidin                                                                    Tampon 1.0        ND         158         10.2       0.09                      w/1.0%                                                                        Lauricidin                                                                    Tampon 1.0        ND         316         10.5       0.08                      w/10.0%                                                                       Lauricidin                                                                    __________________________________________________________________________     ND = NOT DETECTED. Detection limit of the ELISA method of Reiser et al is     0.5 ng/ml.                                                                    .sup.a) = As determined by the ELISA method reported by Reiser et al. in      Applied and Environmental Microbiology, December 1982, pp. 1349-1355, the     disclosure of which is hereby incorporated by reference.                      .sup.b) = Expressed as log to base 10.                                        All data above are mean determinations of duplicate samples.             

The data in Table 1 show that S. aureus bacteria in the presence of atampon comprising 0.1% (w/w) Lauricidin produce 51% less toxic shocksyndrome toxin-1 (TSST-1) than when exposed to a control tamponcontaining no Lauricidin, in spite of the fact that there was noreduction in the actual number of viable S. aureus cells in the presenceof the Lauricidin-treated tampon. The data further show that S. aureusbacteria in the presence of tampons comprising 1.0% (w/w) and 10.0%(w/w) of Lauricidin produced 99% less TSST-1 than did the same number ofS. aureus cells in the presence of a control tampon containing noLauricidin, again in spite of the fact that there was no reduction inthe number of viable S. aureus cells in the presence ofLauricidin-containing tampons. The data show that although the number ofviable S. aureus cells does not decrease when the cells are exposed totampons containing various levels of Lauricidin, the actual amount ofTSST-1 produced by those cells is significantly reduced (as in the caseof the tampon treated with 0.1% Lauricidin) or substantially eliminated(as in the case of tampons treated with 1.0% and 10% of Lauricidin). Inother words, while Lauricidin does not significantly reduce the numberof viable S. aureus bacteria cells, but it does significantly inhibitthe production of toxic shock syndrome toxin-1 by those cells.

Thus, it is believed that in vivo use of Lauricidin treated tampons inmenstruating women would be beneficial in that the production of TSST-1by any S. aureus bacteria normally present in the vagina would besignificantly reduced. In addition, in preliminary studies conductedusing the predominant microorganism present in the vagina, i.e.Lactobacillus acidophilus, it was found that said microorganism was notadversely affected when brought into contact with an Example 1 tamponcomprising 1.0% (w/w) Lauricidin.

EXAMPLE 2

A second experiment was conducted to evaluate growth of, and TSST-1production by, S. aureus cells in the presence of commercially availabletampons treated with varying amounts of Lauricidin. Tampax* brandmenstrual tampons (regular size, Lot No. 8L015Z) which had beenpurchased on the open market were employed in the experiment of thisExample 2. These tampons were manufactured by Tambrands, Inc., LakeSuccess, N.Y., U.S.A. The Tampax* tampons comprised a cotton absorbentcore, a rayon fabric covering, and a withdrawal string. The tamponwithdrawal strings were cut from the tampons prior to testing. Tamponscomprising 0.1%, 1.0% and 10% Lauricidin based on the weight of theuntreated tampons were prepared in duplicate according to theaforementioned General Procedure. Two Tampax* tampons (with strings cutoff), treated with isopropyl alcohol containing no Lauricidin andthereafter dried, were used as a control. The dried, Lauricidin-treatedtampons and the control tampons, were then tested according to theprocedure and conditions reported in Example 1. The test results arereported in Table 2.

                  TABLE 2                                                         ______________________________________                                        THE EFFECT OF LAURICIDIN TREATED TAMPAX                                       TAMPONS (LOT NO. 8L015Z) ON TSST-1 FORMATION                                  BY STAPHYLOCOCCUS AUREUS (FRI-1169)                                                       FINAL                                                                         CONCEN-    FINAL                                                              TRATION    CONCEN-    FINAL                                                   OF S. AUREUS                                                                             TRATION    CONCEN-                                                 CELLS      OF S. AUREUS                                                                             TRATION                                                 (CFU/ml)   CELLS.sup.b)                                                                             OF TSST-1.sup.a)                            SAMPLE      (10.sup.8) (/ml)      (μg/ml)                                  ______________________________________                                        0% Lauricidin                                                                             5,248      11.72      27.50                                       Tampon (control)                                                              0.1% Lauricidin                                                                             199      10.30      3.70                                        On Tampon                                                                     1.0% Lauricidin                                                                             251      10.40      1.05                                        On Tampon                                                                     10.0% Lauricidin                                                                          2.13       8.33       0.19                                        On Tampon                                                                     ______________________________________                                         .sup.a) = As determined by ELISA method (Reiser et al.).                      .sup.b) = Expressed as log to base 10.                                        All sample determinations were made after 24 hrs. incubation at 37.degree     C.                                                                            All data above are mean determinations of duplicate samples.             

The data in Table 2 show that S. aureus bacteria in the presence ofTampax* brand tampons treated with varying amounts of Lauricidin produceless TSST-1 than when exposed to the control tampon containing noLauricidin, the extent of the reduction in toxin production beingrelated to the amount of Lauricidin in the tampons. Tampax* tamponstreated with 0.1% by weight of Lauricidin resulted in an 86% reductionin TSST-1 produced compared to the control, while the Tampax* tamponstreated with 1% and 10% by weight of Lauricidin resulted in,respectively, a 96% and 99% reduction. The results with the Tampax*brand tampons also show a reduction in the total number of S. aureuscells; this effect is dependent upon the concentration of Lauricidin inthe tampon. At the end of the 24 hour incubation period, the logconcentration of S. aureus cells in the presence of the control tamponwas 11.72; the log concentration of S. aureus cells in the presence ofthe tampon containing 0.1% Lauricidin was 10.30 (12% less); the logconcentration of S. aureus cells in the presence of the tamponcontaining 1.0% Lauricidin was 10.40 (11% less); and the logconcentration of S. aureus cells in the presence of the tamponcontaining 10% Lauricidin was 8.33 (29% less).

EXAMPLE 3

A third experiment was conducted to evaluate growth of, and TSST-1production by, S. aureus cells in the presence of commercially availabletampons treated with varying amounts of Lauricidin. Playtex* brandmenstrual tampons (regular size, Lot No. 3496P) which had been purchasedon the open market were employed in the experiment of this Example 3.These tampons were manufactured by International Playtex Inc., Dover,Del., U.S.A. The Playtex* tampons were made of all rayon fiber and had awithdrawal string but no cover fabric. The tampon withdrawal stringswere cut from the tampons prior to testing. Treated tampons comprising0.1%, 1.0% and 10% Lauricidin based on the weight of the untreatedtampon were prepared in duplicate according to the aforementionedGeneral Procedure. Two Playtex* tampons (with strings cut off) withoutany Lauricidin treatment were used as a control. The driedLauricidin-treated tampons and the untreated controls were then testedaccording to the procedure and conditions described in Example 1. Thetest results are reported in Table 3.

                  TABLE 3                                                         ______________________________________                                        THE EFFECT OF LAURICIDIN TREATED PLAYTEX                                      TAMPONS (LOT NO. 3496P) ON TSST-1 FORMATION                                   BY STAPHYLOCOCCUS AUREUS (FRI-1169)                                                       FINAL                                                                         CONCEN-    FINAL                                                              TRATION    CONCEN-    FINAL                                                   OF S. AUREUS                                                                             TRATION    CONCEN-                                                 CELLS      OF S. AUREUS                                                                             TRATION                                                 (CFU/ml)   CELLS.sup.b)                                                                             OF TSST-1.sup.a)                            SAMPLE      (10.sup.8) (/ml)      (μg/ml)                                  ______________________________________                                        0% Lauricidin                                                                             3,388      11.53      10.86                                       Tampon (control)                                                              0.1% Lauricidin                                                                           213        10.33      2.69                                        On Tampon                                                                     1.0% Lauricidin                                                                           131        10.12      0.38                                        On Tampon                                                                     10.0% Lauricidin                                                                          3.23       8.51       0.29                                        On Tampon                                                                     ______________________________________                                         .sup.a) = As determined by ELISA method (Reiser et al.).                      .sup.b) = Expressed as log to base 10.                                        All sample determinations were made after 24 hrs. incubation at 37.degree     C.                                                                            All data above are mean determinations of duplicate samples.             

The data reported in Table 3 show that the amount of TSST-1 produced byS. aureus bacteria in the presence of Playtex* tampons treated with0.1%, 1.0% and 10.0% w/w Lauricidin was reduced 75%, 96% and 97%,respectively, when compared to the amount of TSST-1 produced in thepresence of a control tampon containing no Lauricidin. On the otherhand, compared to control values, the Playtex* tampons treated with0.1%, 1.0% and 10.0% w/w of Lauricidin resulted in 10%, 12% and 26%fewer S. aureus cells at the end of the 24 hour incubation period.

EXAMPLE 4

A fourth experiment was conducted to evaluate growth of, and TSST-1production by S. aureus cells in the presence of another brand ofmenstrual tampons treated with varying amounts of Lauricidin. Rely*brand menstrual tampons (regular size, Lot No. 2060LC01A) which had beenpurchased prior to September 1980 on the open market were employed inthe experiment of this Example 4. These tampons were manufactured byProcter & Gamble, Cincinnati, Ohio, USA. The Rely* tampons comprisedcarboxymethyl cellulose dispersed in a polyester foam which was wrappedin a nonwoven fabric made of spunbonded polyester fibers. They had theusual withdrawal strings. The tampon withdrawal strings were cut fromthe tampons prior to testing. Treated tampons comprising 0.1%, 1.0% and10% Lauricidin based on the weight of the untreated tampon were preparedin duplicate according to the aforementioned General Procedure. TwoRely* tampons (with their withdrawal strings cut off) without anyLauricidin treatment were used as a control. The driedLauricidin-treated tampons and the untreated control tampons were thentested according to the procedure and conditions described in Example 1.The test results are reported in Table 4.

                  TABLE 4                                                         ______________________________________                                        THE EFFECT OF LAURICIDIN TREATED RELY                                         TAMPONS (LOT NO. 2060LC01A) ON TSST-1                                         FORMATION BY STAPHYLOCOCCUS AUREUS (FRI-1169)                                             FINAL                                                                         CONCEN-    FINAL                                                              TRATION    CONCEN-    FINAL                                                   OF S. AUREUS                                                                             TRATION    CONCEN-                                                 CELLS      OF S. AUREUS                                                                             TRATION                                                 (CFU/ml)   CELLS.sup.b)                                                                             OF TSST-1.sup.a)                            SAMPLE      (10.sup.8) (/ml)      (μg/ml)                                  ______________________________________                                        0% Lauricidin                                                                             12,303     12.09      64.32                                       Tampon (control)                                                              0.1% Lauricidin                                                                            2,818     11.45      6.92                                        On Tampon                                                                     1.0% Lauricidin                                                                            1,995     11.30      1.54                                        On Tampon                                                                     10.0% Lauricidin                                                                           1,096     11.04      0.09                                        On Tampon                                                                     ______________________________________                                         .sup.a) = As determined by ELISA method (Reiser et al.).                      .sup.b) = Expressed as log to base 10.                                        All sample determinations were made after 24 hrs. incubation at 37.degree     C.                                                                            All data above are mean determinations of duplicate samples.             

The data reported in Table 4 show that the amount of TSST-1 produced byS. aureus bacteria in the presence of the Rely* tampons treated with0.1%, 1.0% and 10.0% w/w Lauricidin was reduced by 89%, 97% and 99%,respectively, when compared to the amount of TSST-1 produced in thepresence of a control tampon containing no Lauricidin. Whereas, at theend of the 24 hour incubation period, the total S. aureus cellconcentration (expressed as log to the base 10) in the presence of thecontrol tampon was 12.09, the total S. aureus cell concentration(expressed as log to the base 10) in the presence of the Rely* tampontreated with 0.1%, 1.0% and 10% Lauricidin was, respectively, 11.45(5.3% less), 11.20 (7.4% less) and 11.04 (7.8% less).

EXAMPLE 5

A fifth experiment was conducted to evaluate growth of, and TSST-1production by S. aureus cells in the presence of commercially availabletampons treated with varying amounts of Lauricidin. O.b.* brandmenstrual tampons (regular size, Lot No. 0694T) which had been purchasedon the open market were employed in the experiment of this Example 5.These tampons were distributed by Personal Products Company, Milltown,N.J., U.S.A. The o.b.* tampons comprise a blend of rayon and cotton.They included a withdrawal string but did not have an outer cover sheet.The tampon withdrawal strings were cut from the tampons prior totesting. Treated tampons comprising 0.1%, 1.0% and 10% Lauricidin basedon the weight of the untreated tampon were prepared in duplicateaccording to the General Procedure described earlier herein. Two o.b.*tampons (with strings cut off) without any Lauricidin treatment wereused as a control. The dried Lauricidin-treated tampons and theuntreated control tampons were then tested according to the procedureand conditions described in Example 1. The test results are reported inTable 5.

                  TABLE 5                                                         ______________________________________                                        THE EFFECT OF LAURICIDIN TREATED O.b.*                                        TAMPONS (LOT NO. 0694T) ON TSST-1 FORMATION                                   BY STAPHYLOCOCCUS AUREUS (FRI-1169)                                                       FINAL                                                                         CONCEN-    FINAL                                                              TRATION    CONCEN-    FINAL                                                   OF S. AUREUS                                                                             TRATION    CONCEN-                                                 CELLS      OF S. AUREUS                                                                             TRATION                                                 (CFU/ml)   CELLS.sup.b)                                                                             OF TSST-1.sup.a)                            SAMPLE      (10.sup.8) (/ml)      (μg/ml)                                  ______________________________________                                        0% Lauricidin                                                                             3,388      11.53      13.46                                       Tampon (control)                                                              0.1% Lauricidin                                                                             158      10.20      3.26                                        On Tampon                                                                     1.0% Lauricidin                                                                             316      10.50      0.28                                        On Tampon                                                                     10.0% Lauricidin                                                                            95       9.98       0.19                                        On Tampon                                                                     ______________________________________                                         .sup.a) = As determined by ELISA method (Reiser et al.).                      .sup.b) = Expressed as log to base 10.                                        All sample determinations were made after 24 hrs. incubation at 37.degree     C.                                                                            All data above are mean determinations of duplicate samples.             

The data presented in Table 5 show that the amount of TSST-1 produced byS. aureus bacteria in the presence of o.b.* tampons comprising 0.1%,1.0% and 10% by weight Lauricidin was reduced by 75%, 98% and 98%,respectively, when compared to the amount of TSST-1 produced in thepresence of a control o.b.* tampon containing no Lauricidin. The totalS. aureus cell concentration (expressed as log to the base 10) in thepresence of the control tampon was 11.53. The total S. aureusconcentration (expressed as log to the base 10) in the presence of theo.b.* tampon treated with 0.1%, 1.0% and 10% by weight of the Lauricidinwas, respectively, 10.20 (11% less), 10.50 (8.9% less), and 9.98 (13%less).

EXAMPLE 6

A sixth experiment was conducted to evaluate growth of, and TSST-1production by S. aureus cells in the presence of commercially availabletampons treated with varying amounts of Lauricidin. Kotex* Security*brand menstrual tampons (regular size, Lot No. 5C0907C) which had beenpurchased on the open market were employed in the experiment of thisExample 6. These tampons were marketed by Kimberly-Clark Corporation,Neenah, Wisc., U.S.A. They comprised a blend of 60% cotton and 40%rayon, had the usual withdrawal string, and were covered with a nonwovenfabric made of polypropylene fibers. The tampon withdrawal strings werecut from the tampons prior to testing. Treated tampons comprising 0.1%,1.0% and 10% Lauricidin based on the weight of the untreated tampon wereprepared in accordance with the above-described General Procedure. TwoKotex* Security* tampons (with strings cut off) without any Lauricidintreatment were used as controls. The dried Lauricidin-treated tamponsand the untreated control tampons were then tested according to theprocedure and conditions described in Example 1. The test results arereported in Table 6.

                  TABLE 6                                                         ______________________________________                                        THE EFFECT OF LAURICIDIN TREATED KOTEX                                        SECURITY TAMPONS, LOT NO. 5C0907C ON TSST-1                                   FORMATION BY STAPHYLOCOCCUS AUREUS (FRI-1169)                                             FINAL                                                                         CONCEN-    FINAL                                                              TRATION    CONCEN-    FINAL                                                   OF S. AUREUS                                                                             TRATION    CONCEN-                                                 CELLS      OF S. AUREUS                                                                             TRATION                                                 (CFU/ml)   CELLS.sup.b)                                                                             OF TSST-1.sup.a)                            SAMPLE      (10.sup.8) (/ml)      (μg/ml)                                  ______________________________________                                        0% Lauricidin                                                                             1,698      11.23      10.19                                       Tampon (control)                                                              0.1% Lauricidin                                                                             194      10.29      4.90                                        On Tampon                                                                     1.0% Lauricidin                                                                             426      10.63      0.09                                        On Tampon                                                                     10.0% Lauricidin                                                                            426      10.63      0.05                                        On Tampon                                                                     ______________________________________                                         .sup.a) = As determined by ELISA method (Reiser et al.).                      .sup.b) = Expressed as log to base 10.                                        All sample determinations were made after 24 hrs. incubation at 37.degree     C.                                                                            All data above are mean determinations of duplicate samples.             

The data presented in Table 6 show that the amount of TSST-1 produced byS. aureus bacteria in the presence of Kotex* tampons comprising 0.1%,1.0% and 10% by weight of Lauricidin was reduced by 52%, 99% and 99%,respectively, when compared to the amount of TSST-1 produced under thesame experimental conditions in the presence of a control Kotex* tamponcontaining no Lauricidin. The total concentration of S. aureus cells(expressed as log to the base 10) in the presence of the Kotex* tamponscontaining 0.1%, 1.0% and 10% by weight of Lauricidin was, respectively,10.29 (8.4% less), 10.63 (5.3% less) and 10.63 (5.3% less).

As can be seen from the preceding Examples 1-6, a variety of tampons,one of which was made by the inventors (Example 1), others of which werecommercially available (Examples 2, 3, 5 and 6) and one of which hadbeen commercially available but was subsequently withdrawn fromcommercial distribution (Example 4), have been treated with varyinglevels of Lauricidin, a commercially available mixture comprising 93% byweight glycerol monolaurate and 3.5% by weight glycerol dilaurate. Thedata reported in Tables 1-6 show that, depending on the levels ofLauricidin in the tampons, S. aureus bacteria produce significantly lessTSST-1 or, in other words, are inhibited from producing significantamounts of TSST-1 when compared to the amounts of TSST-1 produced, underthe same experimental conditions, by S. aureus bacteria in the presenceof control tampons containing no Lauricidin.

EXAMPLE 7

Test tampons of the kind used in Example I were used in this Example 7.Test tampons comprising 0.1%, 0.5%, and 1.0% Lauricidin based on theweight of the untreated test tampons were prepared according to theGeneral Procedure described earlier herein and were tested according tothe Tampon Sac Method described in Example 1. In this Example 7,however, the tampon sacs were inoculated with different strains of S.aureus bacteria prior to the insertion therein of the Lauricidin treatedtampons. The respective strains of S. aureus tested are identified inTable 7. The concentration of S. aureus at the outset of the experimentwas 1×10⁸ CFU/ml. TSST-1 producing S. aureus strain FRI-1169 utilized inthis Example 7 was obtained from Merlin Bergdoll, Ph.D., Food ResearchInstitute, University of Wisconsin, Madison, Wisc. U.S.A. TSST-1producing S. aureus strain designated 1169W was obtained from FredQuimby, V. M. D., Ph.D., Cornell Medical School, New York, N.Y., U.S.A.A third S. aureus strain (specifically a substrain of FRI-1169) wasisolated from the parent strain and designated TSS Isolate. This TSSIsolate can be obtained in a lyophilized state from S. K. Brown-Skrobot,Ph.D., Personal Products Company, Milltown, N.J., U.S.A. A fourth TSST-1producing S. aureus strain designated Mn8, was obtained from PatrickSchlievert, Ph.D., University of Minnesota, Minneapolis-St. Paul, Minn.,U.S.A. A fifth TSST-1 producing S. aureus strain designated 1187 wasobtained from Keith T. Holland, Ph.D., University of Leeds, Leeds,England. All of the S. aureus strains tested in this example can beobtained from the aforementioned individuals.

Suspensions of the various strains were prepared as described in Example1 and used to inoculate the sacs prior to insertion of the tampons whichwere then tested according to the Tampon Sac Method described inExample 1. Duplicate test tampons without any Lauricidin were used ascontrols. The test results are reported in Table 7.

The results shown in Table 7 show a reduction in TSST-1 formation withincreasing concentration of Lauricidin. This was noted in all fivestrains which were tested. It was concluded from the test results thatthe beneficial effects of Lauricidin observed in Examples 1-6 were notspecific to any particular TSST-1 producing S. aureus strain.

                  TABLE 7                                                         ______________________________________                                        THE EFFECT OF LAURICIDIN TREATED TAMPONS                                      ON GROWTH OF AND TSST-1 PRODUCTION BY                                         VARIOUS STRAINS OF S. AUREUS                                                              CONCENTRATION  TOTAL AMOUNT                                       TSST-1      OF             TSST-1                                             PRODUCING   LAURICIDIN.sup.a)                                                                            PRODUCED.sup.b)                                    STRAIN      (%)            (μg)                                            ______________________________________                                        1169W       NONE           56.54                                              (Quimby Strain)                                                                           0.1            17.54                                                          0.5            0.35                                                           1.0            0.07                                               FRI-1169    NONE           48.75                                              (Bergdoll Strain)                                                                         0.1            5.06                                                           0.5            0.04                                                           1.0            0.03                                               TSS Isolate NONE           53.04                                              (Substrain of                                                                             0.1            5.25                                               FRI-1169)   0.5            1.27                                                           1.0            0.48                                               Mn8         NONE           66.30                                              (Schlievert 0.1            0.66                                               Strain)     0.5            0.12                                                           1.0            0.05                                               1187        NONE           46.80                                              (Holland Strain)                                                                          0.1            6.63                                                           0.5            0.92                                                           1.0            0.58                                               ______________________________________                                         .sup.a) = based on weight of untreated tampon.                                .sup.b) = Total TSST1 per tampon after 24 hr. incubation at 37° C.     All samples were tested for total TSST1 produced using the ELISA method       (Reiser et al.).                                                         

EXAMPLE 8

In this Example 8, tampons comprising various fatty acid esters weretested to determine their effect on growth of and TSST-1 formation by S.aureus bacteria (FRI-1169). Test tampons of the kind used in Example 1were used for this Example 8. All of the test tampons weighed 2.6 grams.0.65 gram of each fatty acid ester to be tested was dissolved in 99.35grams of reagent grade or ester mixture isopropyl alcohol. Four (4)grams of each fatty acid ester solution were applied to the outersurfaces of each of two test tampons to provide treated tamponscomprising 1% by weight of the ester or ester mixture based on theweight of the untreated test tampon. The alcohol was removed byevaporation at 70° C., after which the treated tampons were testedaccording to the Tampon Sac Method described in Example 1. Following isa list of the fatty acid esters which were evaluated:

Tampon No. 1--A mixture of glyceryl monocaprylate and glyceryl caprate.Caprylic acid is a saturated fatty acid containing 8 carbon atoms.Captic acid is a saturated fatty acid containing 10 carbon atoms. Themixture contained about 38.3% by weight of the caprylate ester, about36.9% by weight of the caprate ester and about 0.6% free glycerine. Theremainder of this mixture contained minor amounts of di- and triestersof the two fatty acids.

Tampon No. 2--Glyceryl monolaurate of 90-95% purity and containing 0.2%free glycerine and minor amounts of the di- and triesters. Lauric acidis a saturated fatty acid containing 12 carbon atoms.

Tampon No. 3--Glyceryl monomyristate of 90-95% purity and containingabout 0.2% free glycerine and minor amounts of the di- and triesters.Myristic acid is a saturated fatty acid containing 14 carbon atoms.

Tampon No. 4--Glyceryl monopalmitate of 90-95% purity and containing0.2% free glycerine and minor amounts of the di- and triesters. Palmiticacid is a saturated fatty acid containing 16 carbon atoms.

Tampon No. 5--Glyceryl monostearate of 90-95% purity and containing 0.2%free glycerine and minor amounts of the di- and triesters. Stearic acidis a saturated fatty acid containing 18 carbon atoms.

Tampon No. 6--Glyceryl monooleate of 90-95% purity and containing 0.2%free glycerine and minor amounts of the di- and triesters. Oleic acid isan unsaturated fatty acid containing 18 carbon atoms and one doublebond.

In this Example 8, two untreated tampons were used as controls.

The results of the tests are shown in Table 8. The data show that therewas a marked reduction in the amount of TSST-1 produced by S. aureusstrain FRI-1169 in the presence of the tampons treated with the variousfatty acid esters when compared to the amount of TSST-1 produced in thepresence of the untreated control tampons. The reduction in the amountof TSST-1 produced ranged from about 90% to 99%, except in the case ofthe tampon containing glyceryl monostearate. The 60% reduction in TSST-1production observed in the case of the tampon containing glycerylmonostearate, though not as high as that obtained with the tamponscontaining other esters, was nevertheless quite substantial and isregarded as significant. No corresponding pattern of reduction in thenumber of viable S. aureus cells was observed. It should be noted,however, that at the end of the 24 hour incubation period, there werefewer viable S. aureus cells on the treated tampons than on the tamponshaving no ester treatment.

                                      TABLE 8                                     __________________________________________________________________________    IMPACT OF VARIOUS GLYCERYL ESTER COMPOUNDS ON GROWTH OF                       AND TSST-1 FORMATION BY STAPHYLOCOCCUS AUREUS (FRI-1169)                              FINAL      FINAL      TOTAL                                                   CONCENTRATION                                                                            CONCENTRATION                                                                            AMOUNT  REDUCTION                                       OF         OF S. AUREUS                                                                             TSST-1  IN TSST-1                                       S. AUREUS CELLS                                                                          CELLS.sup.b)                                                                             PRODUCED.sup.a)                                                                       FORMATION                               SAMPLE  (CFU/ml)   (/ml)      (ug)    (%)                                     __________________________________________________________________________    Control 4.20 × 10.sup.9                                                                    9.62       17.15   --                                      Tampon                                                                        Tampon No. 1                                                                          5.80 × 10.sup.6                                                                    6.76       0.18    98.9                                    Tampon No. 2                                                                          6.56 × 10.sup.6                                                                    6.81       0.14    99.1                                    Tampon No. 3                                                                          5.04 × 10.sup.8                                                                    8.83       0.58    96.5                                    Tampon No. 4                                                                          1.44 × 10.sup.9                                                                    9.15       1.75    89.7                                    Tampon No. 5                                                                          6.08 × 10.sup.8                                                                    8.78       6.43    60.2                                    Tampon No. 6                                                                          6.86 × 10.sup.8                                                                    8.83       1.01    94.0                                    __________________________________________________________________________     .sup.a) As determined by ELISA method (Reiser et al.)                         .sup.b) Log to the base 10.                                                   All determinations were made after 24 hours at 37° C.                  All data above are mean determinations of duplicate samples.             

EXAMPLE 9

The mixture of glyceryl monolaurate and glyceryl dilaurate used in theexperiments reported in Examples 1-7 hereof was obtained fromLauricidin, Inc. under the tradename of Lauricidin. As indicated earlierherein, this mixture was analyzed and found to contain 93% by weight ofglyceryl monolaurate and 3.5% by weight of glyceryl dilaurate. Mixturesof glyceryl esters of lauric acid were obtained from two other sources.One such mixture was obtained from Stepan Chemical Company, Maywood,N.J. U.S.A. under the tradename Kessco. This mixture was analyzed andfound to contain 50% by weight of glyceryl monolaurate, and 37% byweight of glyceryl dilaurate. Another such mixture was obtained fromHenkel Corporation under the name Monomuls 90-L12 and found to contain96% by weight of glyceryl monolaurate. No glyceryl dilaurate wasdetected. Using the aforementioned General Procedure and the same testtampons as those used in Example 1, the following tampons were preparedin duplicate:

Tampons comprising, respectively, 0.1%, 0.5% and 1.0% of Lauricidinbased on the weight of the untreated test tampons;

Tampons comprising, respectively, 0.1%, 0.5% and 1.0% of the Kesscoester mixture based on the weight of the untreated test tampons; and

Tampons comprising, respectively, 0.1%, 0.5% and 1.0% of Monomuls 90-L12mixture based on the weight of the untreated test tampon.

Tampons treated with isopropyl alcohol without any ester therein wereemployed as controls. All samples were prepared and tested in duplicateaccording to the Tampon Sac Method described earlier herein. Testresults are set forth in Table 9.

                                      TABLE 9                                     __________________________________________________________________________    IMPACT OF VARIOUS GLYCEROL MONOLAURATES                                       ON GROWTH OF AND TSST-1 FORMATION                                             BY STAPHYLOCOCCUS AUREUS (FRI-1169)                                                        FINAL      FINAL      TOTAL                                                   CONCENTRATION                                                                            CONCENTRATION                                                                            AMOUNT  REDUCTION                                       OF S. AUREUS                                                                             OF S. AUREUS                                                                             TSST-1  IN TSST-1                                 %     CELLS      CELLS.sup.b)                                                                             PRODUCED.sup.a)                                                                       FORMATION                          SAMPLE ADD-ON                                                                              (CFU/ml)   (/ml)      (ug)    (%)                                __________________________________________________________________________    Control                                                                              0.0    8.8 × 10.sup.7                                                                    7.94       69.94   --                                 Kessco 0.1   1.28 × 10.sup.8                                                                    8.10       45.24   35                                 Glyceryl                                                                             0.5   4.16 × 10.sup.7                                                                    7.62       33.60   52                                 Monolaurate                                                                          1.0   3.44 × 10.sup.7                                                                    7.53       1.40    98                                 Lauricidin*                                                                          0.1    1.4 × 10.sup.7                                                                    7.15       4.49    93                                 Glyceryl                                                                             0.5    1.3 × 10.sup.7                                                                    7.11       3.93    94                                 Monolaurate                                                                          1.0    3.2 × 10.sup.7                                                                    7.50       0.39    99                                 Monomuls                                                                             0.1   1.28 × 10.sup.8                                                                    8.10       2.41    96                                 90-L12 0.5   4.16 × 10.sup.7                                                                    7.62       0.36    99                                        1.0   3.44 × 10.sup.7                                                                    7.53       0.16    99                                 __________________________________________________________________________     Kessco* contained 50% by weight of glyceryl monolaurate.                      Lauricidin* contained 93% by weight of glyceryl monolaurate.                  Monomuls 90L12 contained 96% by weight of glyceryl monolaurate.               .sup.a) As determined by ELISA method (Reiser et al.)                         .sup.b) Log to the base 10.                                                   All determinations were made after 24 hours at 37° C.                  All data above are mean determinations of duplicate samples.             

It can be seen from the test data set forth in Table 9 that, for anygiven concentration (i.e. add-ons of 0.1%, 0.5% or 1.0% w/w) of estermixture in the tampon, the final amount of TSST-1 produced under thedescribed test conditions is inversely proportional to the concentrationof glyceryl monolaurate in the ester mixture. Thus, for example, wherethe amount of ester mixture in the test tampons was held constant at the0.5% add-on level, the final amounts of TSST-1 declined from 33.60 ugwhen the ester mixture contained 50% by weight of glyceryl monolaurate(i.e. Kessco*), to 3.93 ug when the ester mixture contained 93% byweight GML (i.e. Lauricidin*), to 0.36 ug where the ester mixturecontained 96% glyceryl monolaurate (i.e. Monomuls* 90-L12). Similarreductions in the final amounts of TSST-1 produced were observed wherethe three ester mixtures were used at 0.1% and 1.0% by weight of thetampon. The results set forth in Table 9 suggest that glycerylmonolaurate (which contains two unreacted hydroxyl groups derived fromglycerol) is more effective in inhibiting production of TSST-1 thanglyceryl dilaurate (which contains a single unreacted hydroxyl groupderived from glycerol).

EXAMPLE 10 In vivo Activity of Glyceryl Monolaurate-Impregnated Tampons

Test tampons were made as follows. Avtex rayon (100%) #SN2587 threedenier was used as the test fiber. The fiber was scoured to remove Tween20 and either left as unfinished or coated with glycerol monolaurate(Henkel Monomuls L-90)(hereinafter referred to as "GML"). The analyticaldetermination of the monolaurate content of the material was 96.0%, 2.0%of the 1-3 diestar and 2.0% unidentified material. The fiber was coatedas follows. Seventy-five pounds of rayon fiber was loaded into a holdingtank and the tank filled with water (120 gallons total). Ammonia (NH₃)(29.4% v/v) was added to the water in the holding tank. The system wasthen heated to 200° F. for 30 minutes. The fiber was then washed withhot water (150° F.) three times, the wash water checked for any residualfoam evidencing the presence of Tween 20. The fiber was then washed withcold water, 60° F.

The fiber was transferred to a centrifuge where it was spun while stillhot for 5 minutes to remove excess water. The 75 lbs. of rayon initiallycontained 54 lbs. of water. The rayon was then opened by hand and placedback into the holding tank. Two restraining plates were placed over thefiber to reduce agitation and minimize foaming. Hot water (10°-200° F.)was added followed by four 5-pound samples of GML, each dissolved infive gallons of 170° F. water. The system was pressurized and heated to190° F. and circulated for 30 mintues. After the system was allowed todrain, the fiber was taken to the centrifuge and spun for 5 minutes. Atthis point, there was 52 lbs. of water (70%) remaining. The temperatureof the outside fiber was 160° F. while the temperature of the insidefibers was 175°-180° F.

The moist fiber was placed in a belt oven, which was heated to about250°-260° F. This heat treatment further opened and dried the rayonfiber. The coated and unfinished fiber were then run through a RandoWebber followed by carding in order to form a workable ribbon from whichto make tampons.

After the fiber was scoured or coated, dried and carded the rayon ribbonwas used in the production of 2.30 g tampons. The fiber was compressedbidirectionally and held in a compression puck for five seconds. Aftercompression, the tampons were placed into 0.62" o.d. applicators. Thetampons were wrapped in cellophane and sealed. Control tampons werelabelled (y) while GML-coated tampons were designated (x). The tamponswere made as follows: Blanks were made by cutting the rayon intosections 2.75" in length by 3.0" in width. Fiber orientation for lengthwas machine-direciton and for width, was cross-direction. The sectionswere either built up or torn down to obtain blank weights of 2.28 g. Therayon section was then hand-rolled and covered. For control blanks, thecover was 0.25 oz. Enka bicomponent fabric (2.75"×4.75"). For GMLblanks, the cover was 0.25 oz. Enka bicomponent fabric coated with 2.4%GML solution. The cover was heat sealed to itself using a hand iron. A8/5 White rayon string, available from Blue Mountain Industries, was cutto 13.0" lengths. The string was pierced through one end of each blankat a distance of 5/8" from the end on the piercing unit and then looped.Each blank was tested for anchor strength by manually pulling the strigafter looping. The blanks were compressed bidirectionally (side-to-side,then end-to-end) and held in the compression puck for 5 seconds.Immediately after compression, the tampons were placed in Reggiethree-piece applicator (0.62" o.d.). The pull string was not knotted.The tampons were wrapped in white cellophane sleeves and sealed.

The compressed tampons were analyzed for determination of theconcentration of GML on the tampon fibers. The average concentration ofGML on the tampons tested was 2.38% w/w.

The in vitro impact of the GML tampons compared to that of theunfinished tampons was evaluated using both the Holland Shake FlaskMethod and the Reiser Tampon Sac Methods for evaluation on TSST-1formation by S. aureus. The Reiser Tampon Sac Method has been describedabove in Examples 1 and 7. The results of the determination of theimpact of the GML (2.38% w/w) coated tampons is set forth in Tables 10and 11. Table 10 shows that greater than 99.9% reductions in TSST-1formation were noted when the tampons were evaluated using the followingHolland Shake Flask Method: two liter triple-baffle flasks wereautoclaved containing 500 ml of Difco Brain Heart Infusion Broth. Aftersterilization, five ml of a 24-hour old culture of S. aureus strainidentified as 1187 was added to the flasks. Either 25.0-gram quantitiesof test material or no material (in the control flasks) were added tothe flasks in duplicate. All flasks were incubated at 37 C with shakingat 160 rpm for 24 hours, at which time TSST-1 concentration and total S.aureus cell count determinations were made. TSST-1 level was determinedusing the ELISA test, while total cell counts were made using StandardPlate Count procedures.

Exposure of the GML and unfinished tampons to S. aureus using the TamponSac Method can be seen in Table 11. Reductions in TSST-1 formationranging from 81.1% in media with blood to 95.9% without aredemonstrated, while the impact on the total number of S. aureus cellswas either none at all in the presence of blood or 9.1% in tubes withoutblood.

The Holland Shake Flask Method showed higher antimicrobial activity withthe shaking and agitation of the system. This in vitro test method wasnot predictive of the in vivo situation. In contrast, the Tampon SacMethod was found to be the preferred in predicting the in vivosituation.

The in vivo evaluation of efficacy was performed as follows. Bothcontrol and GML tampons were mailed to the Southwest Research Institutein San Antonio, Tex., for evaluation for reduction to TSST-1 formationby S. aureus in the baboon vagina. Twelve female baboons were identifiedby immobilization with Ketamine HCl and vaginal examination for grossevidence of infection.

Unfinished control tampons had 5 ml of a S. aureus toxigenic straingrown in Brain Heart Infusion Broth for 24 hours at 37° C. absorbed ontotheir distal ends (ends distant from the string). Pre-weighed seedtampons were immediately introduced into the baboon vagina, without theuse of a speculum, and the pull string cut. Rectal temperature andindirect systolic blood pressure was taken and recorded. Five milliliterblood samples were taken from the cephalic vein and the serum stored at-70° C. until analysis for the presence of anti-TSST-1 antibody andclinical chemistry could be executed.

Seed tampons were maintained intravaginally for twelve hours. After thefirst twelve hours, the baboon was immobilized with Ketamine HCl and thetampon was removed. The seed tampon was placed in a pre-weighed 4-oz.plastic cup. Tampon plus cup were weighed and the amount of tamponassociated fluid was calculated. The tampon was transferred to astomacher bag containing 50 ml of sterile saline (0.9% NaCl) and mixedfor 60 seconds. The stomacher fluid was then submitted for quantitativedetermination of total S. aureus cell count and TSST-1 concentration.The total cell count determinations were made using standard plate countmethod and TSST-1 concentrations were determined using radioimmunoassay(R.I.A.).

All tampons inserted after the seed tampon were treated as previouslydescribed. After removal of the seed tampon, all baboons had control (y)tampons inserted intravaginally to allow for additional growth of S.aureus and TSST-1 production within the vaginal cavity. After twelvehours' additional incubation, the animals were divided in two sets ofsix whereby six baboons were tested with the control tampons and sixwith GML coated tampons. After the 48-hour exposure period, all animalshad tampons inserted supplemented with 5.0 ml of their own blood serumbecause of diminished menstrual flow. Total viable S. aureus cell countand TSST-1 level determination were made on all tampons. After alltampons were processed and both toxin and cell count determinations weremade, four animals were excluded from the study (two control animals andtwo GML test animals). In these animals, either the organism was nottransferred to the vaginal cavity to initiate an infection, or lesstoxin or cell levels than those known to have been applied to the seedtampon were found. Table 12 sets forth the data of total toxin permilliliter of tampon associated fluid and total toxin on the tamponswith the impact on cell count. Tha data shown is representative of fouranimals in each test group.

The data set forth in Table 12 demonstrate considerable decreases intoxin formation in the four animals wearing the GML tampons over thosewearing the control tampons alone. FIGS. 1-4 represent the data of theimpact of the GML tampons. Although initially the toxin level in thetest animals wearing control tampons had higher TSST-1 levels, the GMLtampons brought the level of toxin down significantly over that observedin the controls.

The data representing the total toxin produced per 10⁶ cells of S.aureus, thus normalizing the data with respect to individual cells,demonstrates the significant reduction in the tampons containing GMLover the control tampons. A trend of increasing toxin in relation tobacterial cells was noted in the control animals after the addition ofblood on the 60-hour tampons. The trend noted in the control animalsappears to be a direct impact on the cells themselves and the growthcurve. This trend was observed in the animals wearing the GML tampons.

The data set forth in Table 13 show a direct comparison of a percentageof the control of toxin on the tampon-associated fluid and total toxinformed in the tampons in comparison with the control tampons.

                  TABLE 10                                                        ______________________________________                                        IMPACT OF GML COATED TAMPONS ON TSST-1                                        FORMATION USING HOLLAND SHAKE FLASK METHOD                                                                   FINAL  TOTAL                                                                  CONC.  AMOUNT                                                  TOTAL          OF VIA-                                                                              OF VIA-                                                 TSST-1   RE-   BLE S. BLE S.                                                  PRO-     DUC-  AUREUS AUREUS                                                  DUCED    TION  CELLS  CELLS                                   SAMPLE MEDIUM   (ug)     (%)   (cfu/ml)                                                                             (cfu)                                   ______________________________________                                        S. aureus                                                                            BHI      81.57    --     2.4 × 10.sup.9                                                                1.20 × 10.sup.12                  Control                                                                              BHI      1.62     --    1.92 × 10.sup.9                                                                9.60 × 10.sup.11                  Tampon                                                                        GML    BHI      <0.001   99.93 <10    <5.00 × 10.sup.3                  (2.38%)                                                                       Tampon                                                                        S. aureus                                                                            Blood*   66.89    --     2.0 × 10.sup.9                                                                1.00 × 10.sup.12                  Control                                                                              Blood    6.23     --    2.40 × 10.sup.9                                                                1.20 × 10.sup.12                  Tampon                                                                        GML    Blood    0.004    99.92 1.92 × 10.sup.4                                                                9.60 × 10.sup.6.sup.              (2.38%)                                                                       Tampon                                                                        ______________________________________                                         *Denotes defribrinated Sheep Blood added to BHI at 1.0% v/v concentration

                  TABLE 11                                                        ______________________________________                                        IMPACT OF GML COATED TAMPONS ON TSST-1                                        FORMATION USING THE REISER TAMPON SAC METHOD                                                                  CONC.  AMOUNT                                                 TOTAL           OF VIA-                                                                              OF VIA-                                                TSST-1          BLE S. BLE S.                                                 PRO-     REDUC- AUREUS AUREUS                                                 DUCED    TION   CELLS  CELLS                                  SAMPLE MEDIUM   (ug)     (%)    (cfu/ml)                                                                             (cfu)                                  ______________________________________                                        S. aureus                                                                            BHI.sup.(a)                                                                            40.00    --     6.0 × 10.sup.8                                                                 4.68 × 10.sup.9                  Control                                                                              BHI      65.06    --     6.0 × 10.sup.8                                                                 4.68 × 10.sup.9                  Tampon                                                                        GML    BHI      2.63     95.9   8.0 × 10.sup.7                                                                 6.24 × 10.sup.8                  Tampon                                                                        S. aureus                                                                            Blood.sup.(b)                                                                          54.27    --     8.4 × 10.sup.8                                                                 6.55 × 10.sup.9                  Control                                                                              Blood    50.55    --     1.5 × 10.sup.7                                                                 1.17 × 10.sup.8                  Tampon                                                                        GML    Blood    9.52     81.1   3.6 × 10.sup.7                                                                 2.80 × 10.sup.8                  Tampon                                                                        ______________________________________                                         .sup.(a) Denotes Brain Heart Infusion Agar.                                   .sup.(b) Denotes defribrineated Sheep Blood added to BHI at 1.0% v/v          concentration.                                                           

EXAMPLE 11

In this Example 11, tampons comprising various fatty acid esters weretested to determine their effect on growth of and TSST-1 formation by S.aureus bacteria (FRI-1169). Test tampons of the kind used in Example 1were used for this Example 11. Test tampons comprising 0.1%, 0.5%, 1.0%and 10% fatty acid ester based on the weight of the untreated testtampons were prepared according to the General Procedure describedearlier herein and were tested according to the Tampon Sac Methoddescribed in Example 1. Duplicate test tampons without any fatty acidester were used as controls. The test results are report in Table 14.

The following is a list of the fatty acid esters which were evaluated:

Tampon No. 1--2-Hydroxy-1-propyl laurate

Tampon No. 2--Diethylene glycol monolaurate

The test results show that there was a marked reduction in the amount ofTSST-1 produced by S. aureus strain FRI-1169 in the presence of thetampons treated with the various fatty acid esters when compared to theamount of TSST-1 produced in the presence of the untreated controltampons. The reduction in the amount of TSST-1 produced ranged fromabout 7% (in the case of 0.1% 2-hydroxy-1-propyl-laurate) to about 99%(in the case of 1.0% 2-hydroxy-1-propyl-laurate). No correspondingpattern of reduction in the number of viable S. aureus cells wasobserved.

EXAMPLE 12 Effect of Dodecyl Glycerol Ether on TSST-1 Toxin

In this Example, tampons containing dodecyl-glycerol-ether were testedto determine their effect on growth and TSST-1 formation of S. aureusbacteria (Strain FRI-1169). This ether derivative of glycerylmonolaurate was obtained from Henkel Corp. and identified as dodecylglycerin ether with a OH⁻ value of 421, acid value of 0.4 and purity of99%. The material was specified to be greater than 90% mono-dodecylcontent.

Test tampons of the kind used in Example 1 were used for this Example12.

Test tampons comprising 0.1, 0.5, and 1.0% fatty acid ether based on theweight of the untreated test tampons were prepared according to theGeneral Procedure described earlier herein and were tested according tothe Tampon Sac Method described in Example 1. Duplicate test tamponswithout any fatty acid ether were used as controls. The test results arereported in Table 14.

The test results show that the amount of TSST-1 produced by S. aureusbacteria in the presence of the tampons treated with 0.1%, 0.5%, and1.0% w/w dodecyl-glycerol ether was reduced 96%, 99% and 99%,respectively, when compared to the amount of TSST-1 produced in thepresence of a control tampon containing no dodecyl-glycerol ether.Whereas, at the end of the 24-hour incubation period, the total S.aureus cell concentration (expressed as log to the base 10) in thepresence of the control tampon was 10.48, the total S. aureus cellconcentration (expressed as log to the base 10) in the presence oftampons treated with 0.1%, 0.5%, and 1.0% w/w dodecyl-glycerol-ether wasrespectively, 8.07 (23% less), 6.72 (36% less), and 8.21 (22% less).

                  TABLE 14                                                        ______________________________________                                        Effect of Dodecyl-glycerol Ether on Growth and TSST-1                         Formation By S. aureus                                                               Conc.    Final Conc.                                                                             Final Conc.                                                                            Total Amount                                      of       of S. aureus                                                                            of S. aureus                                                                           TSST-1                                            GML      Cells     Cells.sup.b                                                                            Produced.sup.a                             Sample (%)      (CFU/ml)  (/ml)    (ug)                                       ______________________________________                                        Control                                                                              0        .sup. 3.00 × 10.sup.10                                                            10.48    66.91                                      Dodecyl                                                                              0.1      1.17 × 10.sup.8                                                                   8.07     2.77                                       glycerol                                                                             0.5      5.26 × 10.sup.6                                                                   6.72     0.13                                       ether  1.0      1.65 × 10.sup.8                                                                   8.21     0.06                                       ______________________________________                                         .sup.a As determined by ELISA (Reiser et al.)                                 .sup.b Log to the base 10                                                     All determinations were made after 24 hours at 37 C. All data above are       mean determinations of duplicate samples.                                

EXAMPLE 13

A series of experiments were executed by Dr. M. E. Melish, Department ofPediatrics, University of Hawaii to determine the effect ofglyceryl-monolaurate-treated cotton tampons on TSST-1 formation within arabbit vagina and subcutaneous implantation.

Sexually mature female New Zealand White rabbits over 7 months of ageweighing between 4 and 5.5 kg were utilized throughout the study.Simulated menses consisting of defibrinated rabbit blood mixed with 2.5gm/dl (grams per deciliter) bovine serum albumin to yield a finalprotein concentration of 8.5 gm/dl, albumin concentration of 3.5 gm/dland hemoglobin concentration of 9-11 gm/dl. Blood was taken from animalswith no detectable anti-TSST-1 titer determined by radioimmuno bindingassay. TSST-1 levels were measured in the plasma and tampon fluids by aliquid phase competitive binding RIA using radioiodianted TSST-1. Thisassay can detect TSST-1 concentrations above 0.6-1.3 ng/ml.

Vaginal Study

Tampons were prepared with 100% cotton fiber and 1/4 oz bicomponentfusible fiber cover (Enka, made by the Enka Company). The cover wascoated with 1.32±0.28% w/w glyceryl monolaurate while the control tamponconsisted by 100% cotton fiber alone. The fibers, after carding, wererolled and enclosed in an Enka bag having the dimensions 1.2"×2.0". Thefiber weight was 1.0 g with the string being 8's/5 rayon white.

Rabbits were anestheized with ketamine 35 mg/kg and xylazine 6 mg/kg. Atampon was inserted through the urogential sinus into the vagina abovethe urethra by means of a plastic applciator. Five ml of fluid ±1 cc ofstaphylococcal suspension (S. aureus strain FRI-1169) was applied to thetampon through the applicator. A string for tampon removal remained atthe vulvar opening. Blood was obtained for baseline studies via themarginal ear vein. After 4 hours, the animal was again anesthetized, thetampon was removed by gentle traction upon the string, and a secondtampon was inserted. Another 5.0 ml of sterile fluid withoutstaphylococci was applied to the tampon and the second tampon wasallowed to remain in place for 4.0 hours and then removed underanesthetic as before. A third tampon was inserted and another 5 ml ofsterile fluid was injected. The tampons (test or glycerylmonolaurate-treated) were allowed to remain in the vagina for 14-16hours to simulate overnight usage. Blood was obtained for TSST-1quantitation, hematogic and clinical chemistry. Upon removal of thetampons, the weight was determined and the fluid was extracted. TSST-1concentration, pH and cfu of staphylococci were measured byradioimmunoassay, pH meter, and plating of dilutions on mannitol saltagar. This model has been described in detial in Melisch, M. E. et al.,"Vaginal tampon model for Toxic Shock Syndrome", Reviews of InfectiousDis. 11:5238-5247, 1989. The results of this example are presented inTable 15.

Results

The results presented in Table 15 show the effect of the control,untreated tampons as compared to glyceryl monolaurate treated tampons onTSST-1 formation. The data show that the glyceryl monolaurate treatedtampons resulted in lower levels of TSST-1 formation tn the vaginas ofrabbits when compared to the untreated control. Further, the effect ofglyceryl monolaurate was most significant in the 16 hour exposure(Tampon #3).

Subcutaneous Study

Under ketamine and xylazine anestheisa, tampons (either treated withglyceryl monolaurate or untreated controls) were inserted into the loosesubcutaneous tissue at the nape of the rabbits' neck. The tampon wasthen immediately moistened with saline and inoculated with live, washedstaphylococci. Following microorganism injection, animals were monitoredfor fever, clinical changes, and had multiple blood sampling for BUN(blood urea nitrogen), creatinine, calcium and triglyceride laboratorydeterminations to determine whether the animals had become ill with TSSillness. Individual rabbits were considered to have TSS illness if theirBUN or creatinine values exceeded 2 standard deviations and calciumvalues were below 2 standard deviations at 24 and/or 48 hours fromuninfected control mean values. At such blood drawing, TSST-1 wasquantitated using radioimmunoassay. TSST-1 was also measured in urine.The sample tampons (control or treated) were sampled repeatedly forTSST-1 content and cfu/ml staphylococci.

Results

The results of the evaluation of the effect of untreated (control)tampons as compared to glyceryl monolaurate-treated tampons in bothTSST-1 formation and viable S. aureus FRI-1169 cells over days in thesubcutaneous infection model are shown in Tables 16 and 17. In TAble 16,x represents the mean value measured, "range" represents the range ofmeasurements and (N) represents the number of animals tested. Table 17represents the number of animals that contracted TSS illness or diedduring the course of the experiment. The results clearly show in Table17 that glyceryl monolaurate treated tampons resulted in lower TSST-1formation. This effect on reduced TSST-1 and viable staphylococcus countwas most pronounced in the 8-hour and 24-hour samples. These earlyvalues are more applicable to vaginal tampon use than subcutaneousinfection. This reduction in TSST-1 formation translated to less TSSillness and reduced death for the rabbits implanted with glycerylmonolaurate treated tampons.

                  TABLE 15                                                        ______________________________________                                        Effect of Glyceryl Monolaurate Treated Tampons                                On TSST-1 Production In Rabbit Vaginal Model                                         Tampons                                                                         1             2        3                                             Tampon   TSST-1        TSST-1   TSST-1                                        Type     (ng)          (ng)     (ng)                                          ______________________________________                                        Cotton   447           68       3448                                          Control                                                                       (Range)  (292-601)     (1-135)  (1915-4981)                                   Treated  113           10       24                                            Cotton                                                                        (Range)   (64-162)     (5-14)    (2-47)                                       ______________________________________                                    

                                      TABLE 16                                    __________________________________________________________________________    EFFECT OF GLYCERYL MONOLAURATE TREATED TAMPONS ON TSST-1                      PRODUCTION AND CELL VIABILITY BY S. aureus (FRI-1169)                         IN THE RABBIT SUBCUTANEOUS MODEL.                                             SUBCUTANEOUS TAMPON INFECTION                                                 7.5 × 10.sup.9 CFU 1169                                                 MEAN TSST-1 AND ORGANISM LEVELS IN TAMPON FLUIDS                                     DAY 1      DAY 2      DAY 3      DAY 4                                 TAMPON TSST-1     TSST-1     TSST-1     TSST-1                                TYPE   NG/ML                                                                              CFU/ML                                                                              NG/ML                                                                              CFU/ML                                                                              NG/ML                                                                              CFU/ML                                                                              NG/ML                                                                              CFU/ML                           __________________________________________________________________________    NON GML                                                                       COTTON                                                                        X      134.4                                                                              1.3 × 10.sup.7                                                                463.6                                                                              3.6 × 10.sup.7                                                                357.2                                                                              1.2 × 10.sup.7                                                                522.0                                                                              4.6 × 10.sup.7             RANGE  34.8-459.5 43.1-3386.0                                                                              45.8-2917.0                                                                              33.8-2146.1                           (N)    (13)       (13)       (11)       (10)                                  GML                                                                           COTTON                                                                        X      22.3 7.5 × 10.sup.6                                                                31.0 3.2 × 10.sup.6                                                                42.5 4.1 × 10.sup.6                                                                64.2 7.2 × 10.sup.7             RANGE  0-81.9     0-161.9    0-358.4    0-258.5                               (N)    (13)       (14)       (14)       (13)                                  __________________________________________________________________________

                  TABLE 17                                                        ______________________________________                                        EFFECT OF GLYCERYL MONOLAURATE TREATED                                        TAMPONS IN THE RABBIT SUBCUTANEOUS MODEL                                      Subcutaneous Tampon Infection                                                 7.5 × 10.sup.9 CFU FRI 1169                                                      Control          Treated                                             ______________________________________                                        Toxinemia   1/15    (6.7%)     5/14 (35.7%)                                   Toxin in   11/13   (84.6%)    12/13 (92.3%)                                   Urine                                                                         TSS Illness                                                                              13/15   (86.7%)     6/14 (42.9%)                                   Death       5/15   (33.3%)     2/14 (14.3%)                                   ______________________________________                                    

It has now been demonstrated that concentrations of glycerol monolauratethat do not have any observable effect on cell growth were still able toblock TSST-1 production in S. aureus. It has also been demonstrated thatGML inhibits Staphylococcal alpha hemolysin production. Dr. RichardNovick and Dr. Steven Projan have used the methods of gene fusionanalysis and Northern and Western blotting techniques to demonstratethat, in the presence of GML, the structural gene for the TSST-1 toxin,the tst gene is not transcribed by the cells. Gene fusion analysis hasalso confirmed that transcription from the hla alpha hemolysin promoteris likewise inhibited by glycerol monolaurate.

Production of TSST-1 in S. aureus has been found to be dependent onseveral genetic and environmental factors. In order for a strain of S.aureus to express TSST-1 it must carry the tst gene encoding thestructural gene for the protein. Approximately 20% of human clinicalisolates have been found to carry the gene (Kreiswirth, B. N., P. M.Schlievert, and R. P. Novick, "Evaluation of coagulase-negativestaphylococci for ability to produce toxic shock syndrome toxin-1", J.Clin. Microbiol. Vol. 25, p. 2028-2029, 1987). Another genetic componentis the staphylococcal agr locus which is required for transcription ofthe tst gene (Kornblum, et al., "Molecular biology of thestaphylococci", VCH Publishers, New York, p. 373-402, 1990).

TSST-1 production has also been reported to be responsive to a number ofenvironmental factors. Production is manifest during post-exponentialgrowth phase of cells in culture and apparently requires the presence ofoxygen and a neutral Ph (Schlievert, et al., "Production ofstaphylococcal pyrogenic exotoxin type C: influence of physical andchemical factors", J. Infect. Disc., Vol 147, p. 236-242, 1983).Expression of TSST-1 may also be subject to catabolite repression.Control of TSST-1 production is therefore similar to that of a largenumber of other staphylococcal exoproteins such as alpha-hemolysin(Kornblum et al., 1990).

Because many of the reported cases of toxic shock syndrome involvetampon use, agents that block TSST-1 production and which can beincorporated into tampons without adversely affecting the vaginal florahave been sought. GML has been found to block TSST-1 production. It hasalso been reported that the surfactant Pluronic L-92 stimulated TSST-1production (A. C. L. Wong. 1987. Factors Affecting Growth of S. aureusand Production of Toxic Shock Syndrome Toxin-1. Thesis for Doctor ofPhilosophy. pp 32-33). Experiments have now been performed thatdemonstrate that GML does specifically inhibit TSST-1 expression, aswell as inhibiting alpha hemolysin production. These experiments alsodemonstrate that GML blocks TSST-1 and alpha hemolysin expression at thelevel of transcription.

SUMMARY OF NORTHERN AND WESTERN BLOT TESTS

Both the Northern and Western blot tests are used to detect biochemicalspecies. The essential difference between a Northern and a Western blotis that a Northern blot is used for the detection of a species of RNAwhile the Western blot is used to detect specific proteins. Northernblots have been used in connection with the compositions and methods ofthis invention in order to detect the MRNA transcripts of the tst gene,the gene that encodes the TSST-1 protein. Western blots have been usedto detect the TSST-1 protein itself.

The original blotting technology was developed to detect similar DNAspecies using DNA probes. The original blotting method was developed byEd Southern and was called the "Southern blot".

Generally, DNA fragments were generated by restriction endonucleasedigestions. These fragments were then separated by electrophoresis on anagarose gel, transferred to a nitrocellulose filter and then hybridizedto specific DNA probes. The probes were usually labelled with aradioactive tracer in order to detect DNA fragments homologous to theprobe. The tests use Xray film to find the radioactive tracer.

In a Northern blot, RNA is isolated from cells, although not necessarilypurified, and the various species of RNA are separatedelectrophoretically on an agarose gel matrix that contains formaldehyde.The formaldehyde is intended to prevent RNA secondary structure fromaltering mobility. The RNA is separated by size, with the smallerspecies moving the most quickly through the gel matrix. The separatedRNA molecules are transferred to a nitrocellulose filter and the RNAfixed to the gel by heating to 80 C in a vacuum. In the Northern blottechnique used herein, the transfer is effected by capillary actionusing a high salt buffer solution. The RNA transcribed from a given geneis of a discreet size or sizes. Therefore, after hybridization with agene-specific probe only one or a few radioactive bands are visible.

After transfer, the blot is hybridized to a probe specific for the genebeing studied. Radiolabelled probes are preferable for use in thistechnique because of their ease of detection and quantitation. Probescan be prepared in a variety of ways, but most require a clone of thegene or gene segment being studied. The probe is hybridized to the blotusing a nitrocellulose filter at a temperature optimal for RNA-DNAhydrogen bonding between complimentary bases: A-U; T-A; G-C; C-G (forDNA-RNA hybridization). The hybridization temperature can vary dependingupon AT vs. GC content of the gene in question and the buffer conditionsused. So wherever there is RNA on the filter that has a sequence capableof base pairing to the DNA probe the radioactive tracer will havehybridized. The signal is detected by using X-ray film which is placedover the blot.

Western blotting also involves electrophoresis however, in the case ofWestern blotting, the proteins are the macromolecules being analyzed andthe detection method involves treatment of the filter with antibodiesspecific for the protein being analyzed. After the antibodies are bound,the filter is treated with a second antibody specific for the first andcontaining an enzyme that can be detected by a histochemical stain.

EXAMPLE 14

The mechanism by which glycerol monolaurate (hereinafter "GML") reducesTSST-1 toxin formation has been studied by R. Novick and S. Projan atthe Public Health Research Institute in New York, N.Y. Their work showedthat GML inhibits the formation of TSST-1 at the level of transcriptionin the cell. Northern blot analyses showed that TSST-1-specific MRNAproduction was inhibited in cultures where TSST-1 production wasinhibited.

S. aureus strain Mn8 was isolated from a patient withmenstrual-associated TSS. Mn8 is a high level producer of TSST-1.Strains FR1169 and FR1187, also TSST-1 producers were obtained from theUniversity of Wisconsin, Food Research Institute. Strain RN7220,carrying pRN6735, is a producer of alpha hemolysin.

Brain Heart Infusion Broth (Difco) ("BHI") was used in all theexperiments described. GML was prepared at 1% weight/volume (10 mg/ml)in 95% ethanol. Cultures used as inocula were grown at 37 C overnightwithout shaking in a 300 ml baffled, side arm, shaker flask (volume 10ml)L followed by addition of 10 ml additional media. The flasks werethen shaken at 240 revolutions per minute for one hour. This process hasbeen found to produce a log phase culture. The culture was thensubcultured by addition of 1 ml of culture into 20 ml total in a sidearm, 300 ml shaker flask. Growth was monitored turbidometrically using aKlett-Sumerson photoelectric colorimeter with a green filter.Turbidostatic experiments were performed in a New Brunswick BioFlofermentor (Model No. C30) by pumping in fresh medium containing BHI orBHI with GML at a replacement rate of greater than twice the log phasegeneration time, 100 minutes. This ensured the maintenance of aconstant, post-exponential culture. GML was added at a concentration of20 ug/ml at the time of subculturing. This time was termed t=0. Tocompensate for degradation of GML by enzymes, additional GML wasprovided at specific time intervals. The cultures were monitored andmeasured for growth of cells. The data show that S. aureus Strain Mn8(both in the presence of GML and without GML within the growth media)grow equally well, showing no inhibition of growth by GML at theconcentration employed. (See FIG. 1A).

A Western blot analysis was performed as follows: SDS-polyacrylamidegels were prepared and run according to the method of Laemmli (Laemmli,U. S., "Cleavage of structural proteins during the assembly of the headof bacteriophage T4", Nature, Vol. 227, p. 680-685), using a BioRad MiniProtean apparatus. Protein was transferred electrophoretically tonitrocellulose filter (Schliecher and Scheull BAS5) and detection ofmaterial reacting with the TSST-1 specific anti-serum was performed asdescribed in Blake, et al., "A rapid, sensitive method of detection ofalkaline phosphatase conjugated anti-antibody on Western blots", Anal.Biochem., Vol. 136, p. 175-179. Hyperimmune rabbit antiserum was raisedagainst TSST-1. Anti-rabbit ICG antibody prepared in goat and conjugatedto alkaline phosphatase was obtained from Sigma Chemical Co. (St. Louis,Mo.).

Western blot analysis showed that while there is no inhibition ofgrowth, TSST-1 production is severely limited in the culture containingGML (See FIG. 2A). Similar results were also noted for two additional S.aureus strains 1169 and 1187. No extracellular TSST-1 could be detectedin the culture.

Because no extracellular TSST-1 could be detected, the intracellularcell proteins were analyzed, as follows. The cells were harvested bycentrifugation, washed, and lysed. When the celllysates were analyzed byWestern blots, no material reacting to anti-TSST-1 antibody was detectedin samples from the GML treated culture. This demonstrates the lack ofTSST-1 within the cells.

A Northern blot analysis was performed in order to determine whether theGML was responsible for preventing the S. aureus cells from actuallycompleting transcription of the structural gene for making TSST-1, thetst gene, as follows: Whole cell RNA extracts were prepared andseparated on formaldehyde-agarose (1.25% weight/volume) gels andtransferred to nitrocellulose according to the method of Kornblum et al.("A rapid method to quantitate non-labeled RNA species in bacterialcells", Gene, Vol 63, p. 75-85, 1988) A probe specific for the tsttranscript was prepared by nick translation (Rigby, P. W. J., M.Dieckmann, C. Rhodes and P. Berg (1975), "Labelling deoxyribonucleicacids in high specific activity in vitro by nick translation with DNApolymerase.", J. Mol Biol. 113:237-251. from a 287 bp HindII-BamHIfragment internal to the tst coding sequence (Blomster-Hautamaa et al.,"The nucleotide and partial amino acid sequence of toxic-shock syndrometoxin-1", J. Biol Chem , Vol 261, p. 15783-15786; Kreiswirth, et al.,"Evaluation of coagulase-negative staphylococci for ability to producetoxic shock syndrome toxin-1", J Clin. Microbiol Vol 25, p. 2028-2029).

Log phase S. aureus cells as previously described were centrifuged,resuspended, and lysed. The samples were separated by electrophoresis ona formaldehyde-agarose gel transferred to nitrocellulose and probed withnick-translated tst DNA. Although a strong tst-specific signal was seenin samples obtained from control cells, no signal was visible in thesamples obtained from the GML-treated cells. Prolonged exposure of theNorthern blot nitrocellulose filter showed no visible trace oftst-specific MRNA visible, indicating a profound inhibition oftranscription of the TSST-1 MRNA. The results of this experiment showedthat tst specific MRNA is produced only in the cells from the culturenot treated with GML. The results of this Northern blot analysis are setforth in FIG. 2B.

Biochemical assays were performed as follows: Beta-lactamase activitywas assayed spectrophotometrically at Ph 5.8 employing a chromogenicsubstrate, nitrocefin. Alpha hemolysin was assayed using an adaptationof the tube dilution method (Bernheimer, et al., "Isolation andcomposition of staphyslococcal alpha toxin", J. Gen. Microbiol., Vol.30, p. 455-468). Three-fold serial dilution of culture supernatants wereprepared in 10 Mm Tris 150 Mm NaCl Ph 7.3 and added to the substrate(0.5% whole, defibrinated rabbit blood). Samples were incubated at 37 Cfor 90 min. and then held at 4 C for 30 min. Hemolytic activity wasmeasured by determining residual turbidity at 640 nm. 50% lysis pointswere determined by interpolation. Activities in hemolytic units areexpressed as the reciprocal of the dilution yielding 50% lysis with Wood46, a Staphylococcus aureus strain which produces only alpha hemolysin,used as a standard.

Cultures of MN8, FRI1169 and FRI187 were grown as described above. GMLwas added at a concentration of 20 ug/ml at the time of subculturing,which is designated at t-0 in these experiments. To compensate fordegradation of GML by lipases produced by S. aureus, additional GML wasprovided at the indicated time points. The results of a typicalexperiment are shown in FIGS. 1 and 2. FIG. 1 shows the growth curves ofMn8 cultures with and without GML. As can be seen, both cultures appearto grow equally well sharing no inhibition of growth by GML at theconcentration employed. The Western blot shown in FIG. 2A depictsculture supernatants taken from the culture at the points indicated inFIG. 1. The Western blot clearly shows that, while there is noinhibition of growth, TSST-1 production is severely limited in theculture containing GML. Similar results were obtained for both FR1169and FR1187, although both required more frequent GML supplementation toobserve inhibition. If additional GML was not provided during the courseof growth, TSST-1 production was delayed but not completely inhibited.Mn8 was also grown in a fermentor turbidostatically in apost-exponential phase of growth. TSST-1 production was inhibited onlyat concentrations of GML over 40 ug/ml as assayed by Western blot. Whenthe medium was shifted from BHI and GML to BHI alone, TSST-1 productionwas fully restored after 100 minutes.

In addition to analyzing cell supernatants, the cells were harvested bycentrifugation, washed and lysed. When the cell lysates weresubsequently analyzed by Western blots, no material reacting with theanti-TSST-1 antibody was detected in samples from the GML-treatedculture. However, a faint signal was seen in lysates of non-treatedcells without GML. Thus, a faint signal represents the TSST-1 precursor,prior to secretion. The lack of even this precursor signal in theGML-treated cells indicates that the inhibition by GML is not at thelevel of secretion--otherwise one would have expected the accumulationof this precursor in the GML treated cells. It, therefore, follows thatTSST-1 is not synthesized in GML-treated cells.

Transcription of the tst gene in GML-inhibited cultures was analyzed byNorthern blot. Cell samples were obtained from the cultures shown inFIG. 1; whole cell RNA was prepared according to the quantitative methodof Kornblum et al. as described above. These samples were separated byelectrophoresis on a formaldehyde-agarose gel, transferred tonitrocellulose and probed with nick translated tst DNA. The results areshown in FIG. 2B. As can be seen from FIG. 2B, tst-specific MRNA isproduced only in the cells from the culture not treated with GML, whileno signal whatsoever is visible in the samples obtained from the GMLtreated cells. Prolonged exposure of the Northern blot nitrocellulosefilter showed no trace of tst specific MRNA visible, thus indicating aprofound inhibition of transcription of the TSST-1 MRNA. This result wasconfirmed using a transcriptional fusion of the TSST-1 promoter sequenceto the structural gene encoding beta-lactamase. In these experiments,beta-lactamase activity is used as a reporter of transcription. Resultsof a typical experiment are shown in FIGS. 3A and 3B.

It has previously been shown that transcription of the tst gene wasunder the control of the Staphylococcal agr operon (accessory generegulator). The agr locus controls the production of a large number ofextracellular and cell wall bound proteins (e.g., alpha hemolysin andcoagulase). Agr strains of S. aureus, both experimentally derived andnaturally occurring, do not produce TSST-1 even when the tst gene isintact on the S. aureus chromosome (Kornblum et al. 1990). It was,therefore, thought possible that GML acts by inhibiting expression ofthe agr operon. This operon consists of two transcription units, RNAIIand RNAIII). In the case of the agr system, it has been shown that the600 nt transcript, RNAIII, is the positive effector for severalagr-regulated genes, tst among them. However, Northern blot analysis ofRNAII and RNAIII transcription, shown in FIG. 2C, revealed that thesynthesis of agr transcripts is not inhibited by GML. Therefore, it isunlikely that GML inhibition of TSST-1 production involves inhibition ofthe Agr activation pathway.

It has also been found that GML inhibits alpha hemolysin production.Strain RN7220 is a producer of alpha hemolysin. It produces neitherbeta-hemolysin nor delta-hemolysin. This strain has had the agr locusreplaced with the tet(M) gene; the agr positive effector, RNAIII, isproduced constitutively in this strain by virtue of a plasmid constructin which the blaz promoter is directing RNAIII transcription. Thisstrain was grown in the same manner as the TSST-1 producers analyzedabove. In these experiments, alpha hemolysin activity was assayed asdescribed above with the results shown in FIG. 4A and 4B. As can beseen, as was the case with TSST-1, alpha hemolysin production wasinhibited by GML even while cell growth remained unaffected. Using asimilar gene fusion approach as described above, a fusion of the hlapromoter to the beta-lacatamase reporter gene, it was shown that alphahemolysin production was also inhibited at the level of transcription(FIGS. 5A and 5B).

Thus, these results show that the surfactant, GML, inhibits theproduction of alphahemolysin as well as TSST-1 by S. aureus atconcentrations that do not affect growth. It was found that GML must becontinuously supplied in order to observe inhibition of TSST-1production. This is probably because the bacteria produce lipasescapable of degrading lipids such as GML. Production of both of theseextracellular virulence factors was further shown to be inhibited at thelevel of transcription by a mechanism that remains to be elucidated.Possibly, the target of GML inhibition may be signal transductionpathways that regulate expression of exoproteins.

Production of exoproteins by S. aureus, including both TSST-1 andalpha-hemolysin, require a functioning agr locus. However, it has beendemonstrated here that expression of the agr transcripts is not affectedby GML. The fact that agr is apparently not directly involved in the GMLeffect may indicate that other signal transduction systems are involvedin TSST-1 and alpha-hemolysin expression. It has recently been shownthat in addition to agr, a separate, temporal signal is required foralpha hemolysin expression in S. aureus (Vandenesch, et al., "A temporalsignal, independent of agr, is required for hla but not spatranscription in Staphylococcus aureus", J. Bacteriol., Vol. 173, p.6313-6320). It is therefore possible that GML acts by interfering withthe transduction of this temporal signal.

The practical significance of these findings, therefore, is thatabsorbent products such as tampons, wound dressings or nasal packingmaterial and the like, which are capable of acting as reservoirs for thecontinuous supply of GML and related compounds, can block the productionof toxins by pathogenic Staphylococci without significantly affectingthe flora normally found in the vaginal cavity, on skin or in the nasalcavity.

What is claimed is:
 1. A method of inhibiting the production of TSST-1toxin in mammals comprising exposing TSST-1 toxin-producingStaphylococcus aureus bacteria to an absorbent product comprising acompound selected from the group consisting of:a) monoesters of apolyhydric aliphatic alcohol and a fatty acid containing from eight toeighteen carbon atoms and wherein said monoester has at least onehydroxyl group associated with its aliphatic alcohol residue; b)diesters of a polyhydric aliphatic alcohol and a fatty acid containingfrom eight to eighteen carbon atoms and wherein said diester has atleast one hydroxyl group associated with its aliphatic alcohol residue;and c) mixtures of said monoesters and diesters, said compound beingpresent in an amount which is effective to inhibit the production oftoxic shock syndrome toxin-1 by Staphylococcus aureus bacteria when saidproduct is exposed to said bacteria.
 2. A method according to claim 1wherein said absorbent is a catemenial tampon.
 3. A method according toclaim 2 wherein said tampon is applied to a menstruating mammal.
 4. Amethod according to claim 3 wherein said tampon is applied to amenstruating mammal in its vagina.
 5. A method according to claim 1wherein said fatty acid is lauric acid.
 6. A method according to claim 1wherein said polyhydric alcohol is glycerol.
 7. A method according toclaim 1 wherein said compound is glyceryl monolaurate.
 8. A methodaccording to claim 1 wherein said absorbent product is insertedsubcutaneously in said mammal.